Poxvirus is one of the most promising vaccine vectors to date. ALVAC, a canary poxvirus, is a member of the avipox virus genus in the Chordopoxvirus family, and has been developed as a vaccine vector for expressing foreign genes. Clear advantages of ALVAC as a vaccine vector include its wide tropism, capability for insertion of large DNA fragments and high immunogenicity, such as inducing a strong T-lymphocyte response. ALVAC-based recombinant vaccines have an excellent safety profile and their effectiveness against a variety of infectious agents has been demonstrated in both animals and humans. ALVAC undergoes abortive replication in mammalian cells. In ALVAC recombinants, the target genes are controlled by early promoters and are expressed before the block in replication. Inoculation of an ALVAC recombinant expressing rabies G glycoprotein into dogs was sufficient to protect against a lethal rabies virus challenge (Taylor, J. et al (1991) Vaccine 9: 190-93). Vaccination of cats with an ALVAC recombinant expressing feline leukemia virus (FeLV) A subtype Env and Gag protein protects against the development of persistent viremia after exposure to FeLV virus (Tartaglia, J. et al (1993) J. Virol. 67(4): 2370-5). ALVAC recombinants expressing HIV-1 Env and Gag-pol have been shown to induce HIV-1 specific antibodies and cytotoxic T-lymphocyte responses in humans (Evans, T. G. et al (1999(J. Infect. Dis. 180(2): 290-8; Girard, M. et al (1997) Virology 232(1): 98-104)). However, high doses of ALVAC are often required to achieve protective immunity. Therefore, there is a need to improve the immunogenicity of ALVAC-based recombinants. Similar to ALVAC, fowlpoxvirus, another member of the avipox virus genus in the Orthopoxvirus family, has also been developed as a vaccine vector. Fowlpoxvirus-based recombinant vaccines have demonstrated their efficacy against various infectious diseases in animals and particularly in poultry.
Semliki Forest virus (SFV), a positive sense single stranded RNA virus, is a member of the alphavirus genus in the Togaviridae family. The genomic RNA (49S) of SFV is 11,442 bp in length and contains a 5′-cap and a 3′-polyadenylated tail. Two-thirds of the genome at the 5′-end encodes nonstructural proteins (nsP) and the remaining one-third at the 3′ end encodes structural proteins (sP). Upon infection of cells, the genomic RNA serves as mRNA to initiate the translation of a nonstructural polyprotein, which is subsequently cleaved into 4 nonstructural proteins termed “nsP1”, “nsP2”, “nsP3” and “nsP4”. These proteins form replication complexes with host factors to initiate viral RNA replication and subgenomic RNA (26S) transcription. The subgenomic RNA, corresponding to the one-third of the genome at the 3′-end, is used as a template for translation of structural proteins, which are not required for viral RNA replication.
SFV has been recently engineered to produce a self-replicating RNA “replicon” by deletion of the structural protein genes (Liljestrom, P. and Garoff, H. (1991) Nat. Biotechnol. 9(12): 1356-61). This self-replicating RNA replicon can replicate in a variety of cell types ranging from insect to mammalian cells and expresses target genes at high levels. Recombinant vaccines based on the SFV replicon have been developed and have shown protective immunity against a variety of pathogens (Berglund, P. et al; Vaccine 17(5): 497-507; Berglund, P. et al (1997) AIDS Res. Hum. Retroviruses 13(17) 1487-95; Nilsson, C. et al (2001) Vaccine 19(25-26): 3526-36; Fleeton, M. N. et al (2001) J. Infect. Dis. 183(9): 1395-8). However, the SFV replicon expression system has limitations. For example, for efficient delivery of SFV replicons in vivo, it is necessary to package SFV replicons into virus particles. Packaging is achieved by co-transfection of cells with SFV and helper replicons, which express the viral capsid and envelope proteins using electroporation (Smerdou, C. and Liljestrom, P. (1999) J. Virol. 73(2): 1092-8). This packaging procedure not only requires the synthesis of RNAs in vitro, but also has not yet been developed for large-scale viral particle production. Furthermore, in most mammalian cells, host macromolecular synthesis is inhibited following the introduction of the alphavirus replicon, leading to cell death by an apoptotic mechanism. This limits the use of these replicons to express foreign proteins by transient expression. This also limits the use of this system for large-scale production of these vectors for therapeutic applications.
U.S. Pat. No. 6,015,686 describes a eukaryotic-layered vector system. In this system, a cDNA vector is used to launch an alphavirus replicon. While this system circumvents the requirement for isolating RNA, it still suffers from poor gene delivery efficiency common to all plasmid vectors. Therefore, it is essential to develop alternatives for efficient delivery of SFV replicons in vivo.